Establishment Of A Method For Nucleic Acid Detection Of Classical Swine Fever Virus Mediated By Cas12a And Cas13a

Classical Swine Fever(Classical Swine Fever,CSF)is a highly contagious disease that spreads almost all over the world,causing great losses to pig breeding.In recent years,due to the frequent occurrence of atypical Classical Swine Fever,mixed infection of other viruses,and vaccine immune failure,which result to CSF has been difficult to be eradicated in China.How to carry out quickly and reliably field detection independent of experimental equipment,so as to achieve the prevention and control of CSF has become an urgent problem to be concerned of the breeding industry and scientific researchers.Now,the discovery of protein cleavage activity of Cas9,Cas12 and Cas13 has aroused interest in developing new nucleic acid detection tools for virus.In the present study,Cas12a and Cas13a-mediated nucleic acid detection system of Classical Swine Fever Virus(CSFV)was established and applied to live vaccine detection instead of clinical samples of CSFV.The main results are as follows:(1)The conserved sequence of CSFV was obtained by alignment analysis and successfully inserted into the standard plasmid of p UC57-CSFV as a template for subsequent detection of CSFV.(2)Polymerase Chain Reaction(PCR)and Recombinase Polymerase Amplification(RPA)were used to amplify the conserved fragments of CSFV,respectively,and a pair of the best specific primers with were obtained,and the sensitivities of the two amplification methods were determined.The results showed that the sensitivity of the fragments amplified by the two amplification methods was 7×10~3copies/μL.(3)A method of detecting CSFV based on CRISPR-Cas12a system was established.Mastering the Single guide RNA(sg RNA)design skills of CRISPR-Cas12a,and screening the optimal sg RNA for CSFV detection.When the reaction temperature was37℃,the reaction time was 30min,and the Cas12a-ss DNA-reporter1 probe was used,the fluorescence value of the reporting system reached the maximum,the specificity was perfect,and did not react with African Classical Swine Fever Virus(p UC57-ASFV)and Porcine Respiratory and Reproductive Syndrome Virus(p UC57-PRRSV)standard plasmids.The sensitivity of the established CRISPR-Cas12a detection system combined with RPA isothermal amplification was as low as 7×10~0copies/μL.In addition,the one-tube method was successfully applied to the detection of live Classical Swine Fever vaccine.(4)A method of detecting CSFV based on CRISPR-Cas13a system is established.The sensitivity of this method was evaluated by combining RPA amplification with CRISPR-Cas13a and gradient dilution of p UC57-CSFV plasmid.The Cas13a-ss RNA-reporter1 probe in the CRISPR-Cas13a detection system was suitable.When the reaction temperature is 37℃and the reaction time is 25min,the fluorescence value this method is the highest and the specifity is good.It does not react with the standard plasmid of p UC57-ASFV and p UC57-PRRSV virus,The sensitivity of CRISPR-Cas13a combined with isothermal amplification RPA two-step detection system can be as low as 7×10~0copies/μL.This system is also suitable for virus detection in live CSF vaccine.To sum up,this study successfully established the nucleic acid detection system of CSFV mediated by Cas12a and Cas13a,and mastered the technical principle of nucleic acid detection of Cas12a and Cas13a,which can realize the design and operation of nucleic acid detection of various viruses.At the same time,it is hoped that the transformation of this system can be realized after solving the false positive problem,and the fast,reliable and low-cost system can be widely used in the detection of clinical samples.